G-protein-coupled receptors (GPCRs) are one of the most successful target proteins for drug discovery research to date. Approximately 50% of current drugs target GPCRs; about 20% of the top 50 best selling drugs target GPCRs; more than $23.5 billion in pharmaceutical sales annually are ascribed to medications that address this target class. (Drews, J., “Drug Discovery: A Historical Perspective” Science 2000, 287, 1960-1963; Ma, P., and Zemmel, R., “Value of Novelty” Nat. Rev. Drug Discov. 2002, v. 1, 571-572.) Forming a super-family of seven trans-membrane-spanning proteins that are expressed in virtually all kinds of tissues, GPCRs are associated with almost every major therapeutic category or disease class, including pain, asthma, inflammation, obesity, cancer, as well as cardiovascular, metabolic, gastrointestinal and central nervous system diseases.
The tremendous significance of drugs targeting GPCRs lies in the physiological roles of GPCRs—as cell-surface receptors responsible for transducing exogenous signals into intracellular response(s). (Haga, T., and Berstein, G., eds., G-Protein-Coupled Receptors, CRC Press, Boca Raton, Fla., 1999.) Signaling through these receptors regulates a wide variety of physiological processes, such as neurotransmission, chemotaxis, inflammation, cell proliferation, cardiac and smooth muscle contractility, as well as visual and chemosensory perception. In addition to the role normal receptors play in modulating physiological processes, GPCR mutations that result in both gain and loss of function are associated with certain human diseases. For example, GPCR polymorphisms have been linked with hypertension, idiopathic cardiomyopathy (endothelin A receptor), autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia (calcium-sensing receptor), follicular maturation arrest and suppression of spermatogenesis (follicle-stimulating hormone receptor), and bronchodilator desensitization and nocturnal asthma (β2-adrenoceptors).
In the human genome there are about 400-700 GPCRs of therapeutic relevance; of these GPCRs, ligands for about 200 have been discovered. (Pierce, K. L. et al., “Seven-Transmembrane Receptors.” Nat. Rev. Mol. Cell Biol. 2002, v. 3, 639-650.) Although there is very little conservation at the amino acid level among GPCR sequences, all GPCRs share certain structural and mechanistic features. Typically, GPCRs are formed of seven-helical trans-membrane-spanning domains (each ˜20-30 amino acids in length) joined by intra- and extra-cellular loops. The spatial organization of these trans-membrane regions, the extra-cellular N-terminus and the extracellular loops, form the binding sites for extra-cellular ligands. The intracellular loops and carboxyl-terminus form the sites of interaction with signal-transducing heterotrimeric G-proteins and other regulatory proteins, such as receptor kinases and arrestins. A wide variety of ligand species, including biogenic amines, peptides and proteins, lipids, nucleotides, excitatory amino acids and ions, small chemical compounds, etc., can activate GPCRs.
The success of GPCRs as drug targets stems from the fact that the binding of natural ligands to their paired GPCR(s) can be moderated using appropriate small molecule drugs. (Ma, P., and Zemmel, R., “Value of Novelty,”Nat. Rev. Drug Discov. 2002, v. 1, 571-572.) Effective engineering of these drugs is, however, critical as aberrant binding to such a physiologically significant target class can lead to serious side effects. Structural data on GPCRs is limited and rational drug design is a significant challenge. Designing drugs that do not bind to non-targeted GPCRs is almost impossible. Currently, selectivity studies are conducted downstream in the drug discovery process—discarding compounds because of adverse binding at this stage makes the drug discovery process both expensive and time consuming. Given these considerations, and the strong possibility that so-called “orphan” GPCRs, recently discovered as a result of the sequencing of the human genome, may be valuable targets (Howard, A. D., et al., “Orphan G-Protein-Coupled Receptors and Natural Ligand Discovery.” TiPS 2001, v. 22, 132-140), there is a strong need for technologies that enable screening against multiple GPCRs simultaneously.
Given the importance of G-protein-coupled receptors as drug targets, a wide range of technologies has been developed to screen compounds against GPCRs. (e.g., Hemmila, I. A., and Hurskainen, P., “Novel Detection Strategies for Drug Discovery,” Drug Discov. Today 2002, 7, S152-S156.) The increased pace of target identification (Venter, J. C., et al. “The Sequences of the Human Genome,” Science 2001, v. 291, 1304-1351; Hopkins, A. L., and Groom, C. R., “The Druggable Genome,” Nat. Rev. Drug Discov. 2002, v. 1, 727-730) and the increasing size of compound libraries continues to drive the development of novel GPCR screening technologies (Schreiber, S. L., “Target-Oriented and Diversity-Oriented Organic Synthesis in Drug Discovery,” Science 2000, v. 287, 1964-1968). These assays can be classified into cell based and GPCR-membrane based assays. Despite the interest and the overwhelming number of current and future GPCR targets, few methods have been described for simultaneously studying multiple GPCRs. Recently, two groups of researchers have suggested that arrays of transiently transfected cell clusters or GPCR transfected cells on barcoded substrates could be used for multiplexed compound screening. (Ziauddin, J., and Sabatini, D. M., “Microarrays of Cells Expressing Defined cDNAs,” Nature 2001, v. 411, 107-110; Beske, O. E., and Goldbard, S., “High-throughput Cell Analysis Using Multiplexed Array Technologies,” Drug Discov. Today 2002, v. 7, s131-s135.)
The value of parallel analysis afforded by DNA microarrays (e.g., Schena, M., et al. “Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray,” Science 1995, v. 270, 467-470) has inspired the development of protein arrays (e.g., Mitchell, P., “A Perspective on Protein Microarrays,” Nat. Biotechnol. 2002, v. 20, 225-229). Beyond the use of protein abundance profiling as an analogue to gene expression profiling, protein arrays offer the possibility of highly parallel investigations of protein-small molecule and protein-protein interactions. (MacBeath, G., and Schreiber, S. L., “Printing Proteins as Microarrays for High-throughput Function Determination,” Science 2000, v. 289, 1760-1763; Schweitzer, B., et al. “Immunoassays with Rolling Circle DNA Amplification: A Versatile Platform for Ultrasensitive Antigen Detection,” Proc. Natl. Acad. Sci. USA 2000, v. 97, 10113-10119; Fang, Y., et al. “Membrane Protein Microarrays,” J. Am. Chem. Soc. 2002, v. 124, 2394-2395; and Fang, Y., et al. “G-Protein-Coupled Receptor Microarrays for Drug Discovery,” Drug Discov. Today, 2003, v. 18, 755-761.)
Although the importance of combinatorial approaches to drug design has been realized, the biological equivalent of combinatorial chemistry—multi-target screening using protein microarrays—has not. For multi-target screening, GPCR microarrays maximize the potential for effective matching of biological target space to chemical ligand space. Although, protein microarrays are naturally suited for testing compounds against multiple proteins simultaneously, some of the fundamental aspects of multiplexed bioassays using protein chips are yet to be fully demonstrated. One such fundamental aspect is a need to produce assay conditions, which can lead to optimal binding profiles of any given target compounds and/or labeled ligands to the numerous receptors in the arrays. Problems due to non-specific binding of labeled ligands to the receptor microspots and the background surface, non-optimal interaction of target compounds and labeled ligands with the receptors in the arrays under the assay conditions, and the lack of general guidelines for assay buffer design and selection have deterred scientists from testing the feasibility of multiplexed binding assays for compound profiling and screening.
For functional assays using GPCR microarrays, one should also keep in mind other special considerations in order to achieve not only optimal binding profiling of labeled ligands and target compounds to the receptors in the arrays, but also to maximize the assay sensitivity for monitoring the agonist-induced activation of the receptors and sequential the activation of the G proteins coupled with the receptors. Hence, guidance for assay buffer design and methods to reduce the background are needed for realization of full potentials of GPCR microarrays for compound profiling and screening using multiplexed binding assays and functional assays.